Sensitive detection of heavy metal stress in aquatic plants by dissolved oxygen-quenched fluorescence/materials movement-induced beam deflection method

Abstract

Sensitive detection of heavy metal (HM) stress in aquatic plants by dissolved oxygen (DO)-quenched fluorescence/materials movement-induced beam deflection method is demonstrated. Egeria densa Planchon and Cu²⁺ were used as a model aquatic plant and HM ion, respectively. Reproducibility and experimental errors of the method were first investigated in a control culture solution only containing 10⁻⁶ M Ru (II) complex (Tris (2,2’-bipyridyl) ruthenium (II) chloride) without addition of any fertilizer and Cu²⁺. Changes of DO concentration (∆DO) and deflection (∆DE) during the monitoring periods were used as parameters to quantitatively evaluate the experimental errors and detection limits. Averages or means ($\overline{\Delta DO }$, $\overline{\Delta DE }$) and standard deviations (σ_∆DO, σ_∆DE) of ∆DO and ∆DE in seven control experiments with different aquatic plants sheets during both the respiration and photosynthesis processes were obtained. Next, DO and deflection at the middle vicinities of the aquatic plant were monitored during 2 h of both respiration and photosynthesis in presence of 10^–10 ~ 10^–6 M Cu²⁺. Experimental results showed that the aquatic plant began to suffer from the HM stress in some extent in presence of 10^–9 M Cu²⁺. When the concentration of Cu²⁺ was higher than 10^–8 M, changing trends of both DO and deflection with time were not reversed during the respiration and photosynthesis, implying that the materials movements in the physiological activities had been altered greatly. It is demonstrated that the method could sensitively detect the HM stress in the aquatic plants given by nM HM ions in culture solution without addition of a fertilizer. Furthermore, detection limits of the method were quantitatively discussed by considering $\overline{\Delta DO }-3$ σ_∆DO and $\overline{\Delta DE }+3$ σ_∆DE as the minimum detectable changes of DO and deflection caused by the HM stress, respectively.

Graphical Abstract

Introduction

It is a serious challenge to keep increasing the agricultural crops to feed the growing population in the era of global climate change and widespread environmental pollutions, which give plants various environmental stress, and thus limit the growth of plants and reduce productivity of agricultural crops [1]. Among the various environmental stress, heavy metal (HM) stress has emerged as one of the most serious threats to the crop production that might become even more prevalent in the coming decades. Some HM elements such as Cu are essential for plants’ growth [2]. However, even for the essential HM elements, excess concentration in soils or water environments can result in toxicity to plants and crops which further gives a serious health threat to human [3, 4]. Therefore, it is important to explore and understand the influence of HM stress on growth of plants.

A lot of methods such as visual observation of the plant growth, field sampling/laboratory analyses of chemical components in plant samples, measurements of CO₂ and/or O₂ concentration changes in a chamber containing plants [4,5,6,7], and chlorophyll fluorescence measurements directly from plants [8, 9] have been used for studies of HM stress in plants. On the other hand, most of the studies on the HM stress in plants are carried out among 1 ppm ~ 1000 ppm or several 10 mM ~ mM of HM ions concentration [10,11,12,13]. Recently, we have proposed a novel method for studying HM stress in the aquatic plants at a concentration of 1 μM of HM ions [14]. This method is based on the simultaneous monitoring of dissolved oxygen (DO)-quenched fluorescence and material movements-induced deflection of a probe beam at vicinities within micrometers from the aquatic plant surface. In the method, a 405-nm probe laser was focused on the vicinity of an aquatic plant in a culture solution containing a fluorescent probe of 10⁻⁶ M Ru (II) complex (Tris (2,2'-bipyridyl) Ru (II) chloride). Fluorescence of the Ru(II) complex at the vicinity was excited; and consequently, quenched by DO. Changes of DO at the vicinity were monitored realtime by analyzing the quenched fluorescence intensities. Meanwhile, deflection of the probe beam, which was mainly generated by the concentration gradients of the CO₂ and O₂ [14, 15], was also monitored in real time. Changing trends of both the DO and deflection signals with time became greatly different when 10^–6 M HM ions such as Cu²⁺, Ni²⁺, or Co²⁺ existed in the culture solution, because the 10^–6 M HM ions had greatly influenced the physiological activities. This method enables a more sensitive approach, allowing real-time monitoring of DO and material movements in situ within micrometers of the plant surface, while conventional methods are only capable of measuring the average spatial and temporal change in either or both CO₂ and O₂. Furthermore, the method allows for distinguishing movement of materials such as O₂ across different organ surfaces such as roots, stems, and leaves of the plant.

Although our previous work has revealed that the aquatic plants suffered from HM stress in the culture solution containing 1 μM of Cu²⁺, Co²⁺, or Ni²⁺, whether the HM ions lower than 1 μM still give the HM stress in the aquatic plant is unknown. Also, it is worthwhile to explore the threshold, i.e., the lowest concentration of the HM ions at which the aquatic plants begin suffering from the HM stress. In addition, the detection limit of the method for studying the HM stress remains to be investigated. Therefore, this work investigates influence of the HM ions on physiological activities of the aquatic plants at concentrations lower than 1 μM. Egeria densa Planchon (E. densa), which is not only a popular aquarium plant easily growing even in tap water with low light but also behaves as an ecosystem engineer by preventing the re-suspension of sediments and controlling the growth of phytoplankton in natural water environments [16], was used as a model aquatic plant. Copper (II) ion, which is an essential trace element [2, 17] and has been widely used in studies of HM stress in plants [18], was used as a model HM ion. The conventional chlorophyll fluorescence measurements [8, 9] of the aquatic plant are carried out too for a comparison with the present method.

Experimental

Figure 1 illustrates the beam deflection/fluorescence detection (A) and chlorophyll fluorescence measurement (B) systems. The beam deflection/fluorescence detection system was same as that reported previously [14, 19]. The detection system was placed in a dark room; and a red–blue LED (output power: 8 W, Luxour, Japan) with wavelength of about 660 nm and 460 nm illuminated the aquatic plants through the dark room window in photosynthetic process. The model plant E. densa was bought from an aquarium shop in Fukuoka city and was cultured in tap water air-bubbled by an air pump, as done in the aquarium shop. Before the deflection/fluorescence experiments, about 3 cm of E. densa was cut and moved in a culture dish (ϕ56 mm × 15 mm) containing 20 mL of 1 mM Ru (II) complex solution either with or without addition of Cu²⁺, as shown in the photo inside Fig. 1A. The culture dish was placed on an X–Y–Z micro-stage (Edmund Optics) for adjusting distance between the focus point of the probe beam and the plant leaf surface. A semiconductor laser probe beam of 405 nm (output power: 3.0 mW, Sigma Koki, Japan) was used as the light source of both the deflection and fluorescence measurement. The laser beam was reflected at a dichromic mirror and then focused to a middle vicinity of a leaf in the aquatic plant because the middle vicinity of the leaf exhibited the maximum changes of both the deflection and DO [14]. Deflection of the laser probe beam was detected by a position sensor consisting of a bi-cell photodiode. Fluorescence transmitted back through the dichromic mirror was monitored by a photomultiplier tube (PMT). A commercial DO/temperature sensor (pyro science GmbH) was also inserted into the culture dish for monitoring DO and temperature change of the bulk culture solution. The monitored temperature, DO, deflection, and fluorescence intensity were concurrently input into a digital multimeter (Texio Technology Corporation, Japan), and recorded in a computer. The monitored fluorescence intensity was transferred to DO concentration at the vicinity with the same calculation method as previous [14, 19].

Fig. 1

Illustration of the deflection/fluorescence detection system (A) and conventional chlorophyll fluorescence measurement system (B)

In the chlorophyll fluorescence measurement system (Fig. 1B), a pulse amplification modulation (PAM) fluorometer (AquaPen AP 110-P, Photon Systems Instruments, Czech Republic) which directly gave the maximum photochemical efficiency (F_v/F_m) [8, 9] of photosystem II (PSII) was used. The PAM fluorometer head was close to the wall of a cubic culture dish (3 cm × 8 cm × 6 cm) filled with the 10^–6 M Ru (II) complex solution either with or without the addition of Cu²⁺, where a bundle of the aquatic plant (about 7 cm in length) was cultured as shown in photos in Fig. 1B. The chlorophyll fluorescence was measured after dark adaption for at least 30 min [20, 21].

Stock solutions of 10^–2 M Ru (II) complex and 10^–2 M Cu²⁺ were prepared by dissolving certain amounts of Tris (2,2'-bipyridyl) Ru (II) chloride and CuSO₄·5H₂O into 100 mL distilled deionized water, respectively. The stock solutions were diluted to desirable concentrations with distilled deionized water, and further mixed according to suitable mixing ratio for preparation of the culture solutions containing 10^–6 M Ru (II) complex and Cu²⁺ with different concentrations. Experiments in the 10^–6 M Ru (II) complex solution without addition of Cu²⁺ were used as controls for investigating HM stress of the Cu²⁺ at different concentrations.

Results and discussion

First, reproducibility and experimental errors of the DO-quenched fluorescence/materials movements-induced beam deflection method was investigated. Deflection signals and DO concentrations at the middle vicinity of the aquatic plant leaf were monitored for 2 h during both the photosynthetic and respiration process in the control solution, i.e., 10^–6 M Ru (II) complex solution without addition of the Cu²⁺. Figure 2 shows typical results of three different aquatic plant sheets in 3 different days. Changing trends of both the deflection and DO concentration with time during the respiration process were reversed to those during the photosynthesis processes. The reversed changing trends with time reflected the reversed materials’ movements across the plant surface during the respiration and photosynthetic processes, i.e., the aquatic plant absorbed O₂ and released CO₂ during the respiration while reversely during the photosynthesis process. Figure 2 also shows that the changing trends of both DO and deflection with time were reproducible for different aquatic plant sheets.

Fig. 2

Monitored results of beam deflection (A, B) and DO changes (C, D) at vicinities of aquatic plants in 10^–6 M Ru(II) complex solution without addition of Cu²⁺ at different days. A, C LED-off; B, D LED-on

It is concerned whether the 10^–6 M Ru (II) complex would give HM stress or toxicities to the aquatic plant. Although excess HM ions are toxic to plants, complexations or chelation of HM ions is known to be one of the important HM detoxification methods in plants [22]. Also, little adsorption or absorbance of the Ru (II) complex in the aquatic plants was found in our preliminary experiments [15], and reverse changing trends of both deflection and DO were observed in the respiration and photosynthesis processes. Therefore, influence of the 10^–6 M Ru (II) complex on the physiological activities of the aquatic plants was small enough to be ignorable in comparison with the HM stress, which altered the changing trends of both the deflection and DO, as reported in the previous [14] and as follows.

Changes of DO concentration (∆DO) and deflection (∆DE) during the monitoring periods were used as parameters to quantitatively evaluate the experimental errors and detection limits. They were calculated as follows.

$$ \Delta {\text{DO }} = {\text{ DO}}_{{\text{e}}} - {\text{ DO}}_{{\text{b}}} $$

(1)

where DO_e and DO_b are DO concentration at the end and beginning of the monitoring period, respectively. Considering noise in measurements, averages of the DO concentrations during the last and first 10 s during the monitoring period were used as the DO_e and DO_b, respectively. Similarly, change of the deflection (∆DE) during the monitoring period was also calculated as follows.

$$ \Delta {\text{DE }} = {\text{ DE}}_{{\text{e}}} - {\text{DE}}_{{\text{b}}} $$

(2)

where DE_e and DE_b are averages of the deflection signals during the last and first 10 s of the monitoring period, respectively.

In analytical science, detection limits of analytes are usually defined as the concentrations or masses at which signal-to-noise ratio is 3, or at which difference between the analytical signal and mean blank signal is triple of standard deviation [23]. Here, averages or means ($\overline{\Delta DO }$, $\overline{\Delta DE }$) and standard deviations (σ_∆DO, σ_∆DE) of ∆DO and ∆DE in seven control experiments with different aquatic plants sheets during both the respiration and photosynthesis processes are first obtained. The σ_∆DO and σ_∆DE reflected the experimental errors and heterogeneities of the aquatic plant sheets. Then Cu²⁺ with different concentrations were added to the control solutions; and values of the ∆DO and ∆DE during both the respiration and photosynthesis process are investigated. When the ∆DO and ∆DE in the presence of Cu²⁺ are equal to $\overline{\Delta DO }+3$ σ_∆DO (or $\overline{\Delta DO }-3$ σ_∆DO) and $\overline{\Delta DE }+3$ σ_∆DE (or $\overline{\Delta DE }-3$ σ_∆DE), respectively, the Cu²⁺ concentration is considered to be the detection limits of the method for detecting the HM stress, and the Cu²⁺ concentration is also the threshold to give the HM stress.

Figure 3 shows monitored results of both the deflection and DO changes with time at middle vicinities of the aquatic plant leaves in presence of Cu²⁺ with different concentrations during both the respiration and photosynthetic processes. In the control experiments without addition of Cu²⁺, changing treads of both the deflection signals and DO with time during the respiration process were reverse to those during the photosynthetic process, as stated above. When the added Cu²⁺ concentration was 10^–10 M, changing trends of both the deflection signals and DO with time were similar as those in the control experiments during both the respiration and photosynthetic processes. This meant that presence of the 10^–10 M Cu²⁺ had not given the HM stress to the aquatic plant. When the added Cu²⁺ concentration was 10^–9 M, changing trends of deflection signals with time became same until about 5200 s during both the respiration and photosynthetic processes. After about 5200 s, deflection signals changed with time during the respiration process reversely to those during the photosynthesis. On the other hand, DO at the vicinities of the plant leaf decreased with time faster than the control experiment during the respiration process, while increased with time slower than those in the control after about 2000s. After about 5200 s, DO decreased with time even during the photosynthesis. This suggests that the physiological activities of the aquatic plant have been altered partially, implying the aquatic plant has suffered from the HM stress to a certain extent in presence of the 10^–9 M Cu²⁺. When the added Cu²⁺ concentration was 10^–8 M and 10^–7 M, changing trends of both deflection and DO with time during the respiration became completely same as those during the photosynthetic processes. The deflection signals increased while DO decreased with time faster in presence of 10^–7 M than 10^–8 M during both the respiration and photosynthesis. This suggests that the physiological activities of the aquatic plant have been altered greater in presence of 10^–7 M than 10^–8 M Cu²⁺; meaning that the 10^–7 M Cu²⁺ gave more HM stress in the aquatic plant than the 10^–8 M Cu²⁺.

Fig. 3

Monitored results of the beam deflection (A, B) and DO (C, D) at vicinities of aquatic plant leaf in respiration (A, C) and photosynthetic (B, D) processes with different concentrations of Cu²⁺

Figure 4 shows the relationship between the added Cu²⁺ concentrations and the ∆DE and ∆DO during both the respiration and photosynthesis process. The means of the $\overline{\Delta DO }$ and $\overline{\Delta DE }$ in the control experiments, values of $\overline{\Delta DE }+3$ σ_∆DE and $\overline{\Delta DO }-3$ σ_∆DO, and the ∆DE and ∆DO at different Cu²⁺ concentrations were presented as squares, dash lines, and circles, respectively. Figure 4A, B shows increasing and decreasing trends of the ∆DE and ∆DO with the added Cu²⁺ concentration during both the respiration and photosynthesis process, respectively. The higher the Cu²⁺ concentration was, the larger the values of the positive ∆DE and negative ∆DO were. These results suggested that the HM stress in the aquatic plants increased with the added Cu²⁺ concentration. When the ∆DE was used as an indicator to evaluate the HM stress, detection limits or threshold concentration of the Cu²⁺ were estimated to be about 10^–9 M and 6 × 10^–9 M in the respiration and photosynthesis processes, respectively, because at which the ∆DE would be the $\overline{\Delta DE }+3$ σ_∆DE as shown by dotted lines in Fig. 4A. On the other hand, when the ∆DO was used as the indicator, detection limits or threshold concentration of the Cu²⁺ were estimated to be 5 × 10^–10 M and 8 × 10^–10 M (see dotted lines in Fig. 4B) in the respiration and photosynthesis processes, respectively.

Fig. 4

Relationship between the ∆DE (A) and ∆DO (B) and the added Cu²⁺ concentrations. Means ($\overline{\Delta DO }$, $\overline{\Delta DE }$) of the ∆DE and ∆DO in the control experiments are represented as a square; and values of the ∆DE and ∆DO in the culture solutions added with Cu²⁺ are represented as cycles. Dash lines express the $\overline{\Delta DE }+3$ σ_∆DE (A) and $\overline{\Delta DO }-3$ σ_∆DO (B), respectively. Dot lines are used for estimating Cu²⁺ concentration at which the ∆DE and ∆DO were $\overline{\Delta DE }+3$ σ_∆DE (A) and $\overline{\Delta DO }-3$ σ_∆DO (B), respectively

As stated in the introduction, Cu is an essential element in plants. Copper in terrestrial plants are supplied from soil where Cu is reported to be from 1 μM ~ 1 nM [17]. This might be one reason why most studies of HM stress in plants are being done with HM ions concentrations of several 10 μM ~ mM or several ppm ~ 1000 ppm. Concentrations of Cu in most of natural water environments should be lower than in soils. Therefore, supplying of Cu from natural water environments to aquatic plants or Cu²⁺ equilibriums between aquatic plants/water should be greatly different from those in terrestrial plants, especially for the E. densa growing in tap waters without any addition of fertilizer. Although concentration of Cu²⁺ in tap water of the laboratory was unknown, it has reported that total Cu determined by atomic absorbance spectroscopy were about 1.0 ppb [24] and 2.3 ppb [25] in tap water of Japan. Considering existence of Cu microparticles in tap water, concentration of free Cu²⁺ is estimated to be lower than 10^–8 M. Concentration of Cu²⁺ in the control solution prepared from the distill deionized water should be lower than the tap water. Therefore, after being moved into the control solution from the tap water, the aquatic plant might be in Cu-deficient state. However, the reverse changing trends of both the deflection and DO in Fig. 2 suggested that the Cu deficiency is not so remarkable to affect the reversed materials’ movements including O₂ across the aquatic plant surface during the respiration and photosynthesis in the control solution. On the other hand, Figs. 3 and 4 show that the addition of the 10^–9 M Cu in the control solution altered changing trends of DO, suggesting that addition of 10^–9 M Cu in the control solution has made Cu²⁺ excess to the aquatic plant. Therefore, it is concluded that the aquatic plant began to suffer from the HM stress when the added Cu²⁺ was about 10^–9 M. As far as we knew, this is the first report on detection of the HM stress in aquatic plants given by HM ions as low as 10^–9 M.

It is well-known that the photosynthesis starts when light is absorbed by the antenna chlorophyll molecules of photosystem II (PSII) [8, 9]. The absorbed light energy is used for photochemistry (photosynthesis), heat dissipation, or chlorophyll fluorescence emission. These three processes are in competition, thus measuring the yield of one process can provide information about the changes in the other two processes [8, 9]. Chlorophyll fluorescence measurements have been widely used to study the physiological performance of plants including effect of HM stress on photosynthesis of plants [13, 26, 27]. It has been reported that HM stress and HM toxicity significantly reduced values of chlorophyll fluorescence parameters such as the maximal photochemical efficiency (F_v/F_m) of PSII [28,29,30]. Here, changes of the F_v/F_m with time were investigated in presence of Cu²⁺ with different concentrations, and the results are shown in Fig. 5A. Figure 5A shows that values of the F_v/F_m at the beginning and end of the monitoring period were about 0.7 and 0.58 ~ 0.68 in the control experiments, respectively. When Cu²⁺ concentration was below 10^–5 M, values of the F_v/F_m at the end of the monitoring period were between 0.58 ~ 0.68 too. When Cu²⁺ concentration was 1.0 × 10^–5 M, the F_v/F_m was about 0.60, close to the bottom values of the control ones. When Cu²⁺ concentrations were 1.0 × 10^–4 and 1.0 × 10^–3 M, the F_v/F_m decreased with time greatly. Moreover, the higher the Cu²⁺ concentration was, the faster the F_v/F_m decreased with time. This meant that the maximum photosynthetic ability of the aquatic plants decreased with the Cu²⁺ concentration. This decrease is caused by the HM stress in plants, as reported in the literatures [28,29,30].

Fig. 5

Changes (A) of the the maximal photochemical efficiency (F_v/F_m) of PSII with time at different Cu²⁺ concentrations and relationships (B) between the ∆(F_v/F_m) and add Cu²⁺ concentrations. In B, values of ∆(F_v/F_m) in presence of the added Cu²⁺ and $\stackrel{-}{\Delta \left({F}_{v}/{F}_{m}\right)}$ in the control solutions are represented by circles and a square, respectively. The $\stackrel{-}{\Delta \left({F}_{v}/{F}_{m}\right)}-$ 3(σ_∆(Fv/Fm)) is expressed as a dash line, and the corresponding Cu²⁺ concentration is estimated by the dot line

Similar to the ∆DO and ∆DE, ∆(F_v/F_m), i.e., difference of the F_v/F_m between the end and beginning of the monitoring period, averages or means ($\stackrel{-}{\Delta \left({F}_{v}/{F}_{m}\right)})$ and standard deviations (σ_∆(Fv/Fm)) of the ∆(F_v/F_m) in the control experiments were calculated. Figure 5B shows the relationship between the ∆(F_v/F_m) and Cu²⁺ concentration. The $\stackrel{-}{\Delta \left({F}_{v}/{F}_{m}\right)}$ is represented as a square; and values of the ∆(F_v/F_m) in presence of Cu²⁺ are expressed as circles. Value of $\stackrel{-}{\Delta \left({F}_{v}/{F}_{m}\right)}-$ 3(σ_∆(Fv/Fm)) is indicated as a dash line. Figure 5B shows that the higher the added Cu²⁺ concentration was, the larger the negative value of the ∆(F_v/F_m) was. Figure 5B also shows that the $\stackrel{-}{\Delta \left({F}_{v}/{F}_{m}\right)}-$ 3(σ_∆(Fv/Fm)) corresponded to Cu²⁺ concentration of about 4 × 10⁵ M (see the dot line), suggesting that detection limits of Cu²⁺ for the HM stress by the chlorophyll fluorescence method were about 4 × 10^–5 M. On the other hand, Figs. 3 and 4 show that the present method could detect the HM stress in the aquatic plant when Cu²⁺ concentration was as low as 10^–9 M, much highly sensitive than the chlorophyll fluorescence method.

As a conclusion, it is demonstrated that the method could detect the HM stress in the aquatic plant in presence of HM ions as low as 10^–9 M in the culture solution without addition of a fertilizer. It should be noted that addition of fertilizers and species and concentrations of nutrients in the culture solution greatly affect the HM stress of Cu²⁺. Because this work was aimed to demonstrate the high sensitivity of the present method in detecting the HM stress to the aquatic plant, no nutrients or fertilizers were added in the culture solution. The effects of the nutrients or fertilizers on the HM stress of Cu²⁺ in the aquatic plants are being studied and will be reported later.